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Abstract

The aim of the project was to identify and functionally characterize novel human proteins that dominantly induce apoptosis upon overexpression. To achieve this, a cell-based high throughput assay was developed. The assay is based on the detection of activated caspase-3 in cells overexpressing proteins tagged C- and N-terminally with YFP. Apoptotic cells were detected by staining with a specific antibody directed against the activated form of caspase-3. The assay was automated and data acquisition was done using a flow cytometer with an integrated 96-well plate reader. A total of 200 proteins have been screened in the assay, out of which five were identified to be significant activators of apoptosis. One of the candidates, Vacuole Membrane Protein 1 (Vmp1), which forms vacuoles in cells and subsequently induces apoptosis when overexpressed, has been functionally characterized in detail. It has been reported that VMP1 mRNA is differentially expressed in cancer, acute pancreatitis and kidney ischemia and that the overexpressed protein is localized to the Endoplasmic reticulum. But the function of this protein and its role in cancer and other diseases was previously unknown. In this study I show that the vacuoles are formed by the Endoplasmic reticulum due to accumulation of overexpressed Vmp1, and that Vmp1 is actually a plasma membrane protein involved in the formation of initial cell-cell contacts. Its function as a cell-cell adhesion protein was confirmed by identifying that Vmp1 interacts with the tight junction protein ZO-1, and that down regulation of Vmp1 induces cell detachment. Further, down regulation of Vmp1 resulted in a massive increase in the invasion potential of kidney cancer cells, which is consistent with the findings that VMP1 mRNA level is significantly lower in kidney metastases compared to primary tumours. Thus, these results are the first to show that Vmp1 is a cell adhesion protein, and that its expression level is a critical determinant of cancer cell invasiveness, metastasis formation and induction of apoptosis. In summary, I have established and applied a high throughput cell-based assay to screen for inducers of apoptosis. Functional characterization of one of the candidates from this screen revealed it to be a disease relevant regulator of cell-cell adhesion. This demonstrates the strength of this high throughput approach in the identification of proteins involved in diseases.